Culicinae Mosquito TRA-2 RNA Interference Technique to Genetically Produce Maleness Population

ABSTRACT

This invention entails a method to make a Tra-2 RNAi kernel sequence, and uses for this kernel sequence. The method includes the steps of amplifying an RNA recognition motive (RRM) DNA sequence from a  Culcinae  mosquito species, to obtain a first RRM DNA sequence of 240 base pairs; then reversely connecting the first RRM DNA sequence with a second RRM DNA sequence via an intron or a linker DNA sequence, in such a way that the second RRM DNA sequence forms an inverted sequence to the first RRM DNA sequence. Transcription of the two DNA sequences produces single strands of mRNA that can bind together to form a double strand hairpin mRNA structure. Other steps include inserting a tetracycline repressible transactivator, and an insect spermatogenesis promoter.

This application is a continuation application of Ser. No. 14/356,978,filed May 8, 2014, which is a national application of PCT/VN2011/000014,filed Dec. 29, 2011, which claims priority from Vietnamese applicationVN 1-2011-00772, filed Mar. 23, 2011. This application claims priorityfrom all these documents, and the entire contents of these documents areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology todevelop a novel method for controlling mosquito populations. Culicinaemosquitoes carry one or more loci of transformant Tra-2 RNAi constructswhich target to mosquito Transformer-2 locus in respective or nonerespective Culicinae mosquitoes. Tra-2 sequences used to assemble Tra-2RNAi recombinant constructs are Tra-2 gene sequences of Culicinaemosquitoes and can be derived from endogenous or exogenous sequences.The Tra-2 RNAi expression is conditional, wherein the expression causinga knockdown effect into the endogenous Tra-2 gene results in mortalityof X (m) chromosome bearing sperms and produces maleness mosquitopopulation in the nature environmental of the species.

BACKGROUND OF THE INVENTION

Nowadays, there are many biological control techniques have beeninvented for controlling insects and plants (Smith and Borstel, 1972.Science. 178. 1164-1174). A method invented to control of insectpopulations is named the “sterile insect technique” SIT. This method isalso known with a different name as “sterile insect release method”SIRM. A model of the SIRM based on population parameters obtained from alarge scale experiment to eradicate melon fly was first developed by Ito(1977. Appl. Ent. Zool. 12:310-312; 1979. Res. Popul. Ecol. 20.216-226).The SIT method creates sterile male insects and releases theminto natural habitats where the males would look for natural females tomate. These females would be sterile or to produce offspring that cannotdevelop up to the harmful stages. When a huge number of sterile malesreleased in a chronic time, it can cause a collapse of the naturalinsect populations or even extinction. However, the insect created bythe SIT method need to be undergone a sexing step to remove females. Thereason is that in many insect species, even sterile females, if beingreleased, they would look for blood meals and may transmit diseases (inmosquitoes) or damage fruits (in med fly). As such, release of thefemale insects must be avoided.

Currently, there are different methods to separate male insects from atotal based on the differences of sexual traits as body sizes oreclosion time. However, systematic errors of those are high and more orless depending on each species, for instance, our data indicated that8-15% females of Aedes aegypti mosquitoes can be misidentified as males(K. P. Hoang, unpublished data). Alternatively, insects can be frozendown on ice to separate females and males. However, this method is toohigh labor costs and can damage small insects as male mosquitoes.

In another approach, X and gamma rays are used to translocate achromosome fragment which carries genes encoding for different colors ofsilk worm male and female eggs (Strunnikov, 1979. Theor. Appl. Genet.55, 17-21; Strunnikov. 1983. Control of silkworm Reproduction,Development and Sex. MIR Publishers. Moscow). However, the mutantstrains created by radiation are usually accompanied with a significantdecline in male mating competitiveness in comparison with its wild typemales. This can result in failures in vector control strategies ifapplying for the release of insect males. Besides, irradiation method isnot specific to the certain target, the radiation not only causes bigmutations in chromosome systems of the target organisms but can also bedangerous for producers. This is also an expensive method with plenty oflimitations. Asburner et al., (1998) disclose a method by introductionof an exogenous DNA fragment into the insect genetic system to createinsect transgenic species (Insect Molecular Biology, 7 (3), 201-213).This approach was lately improved by a patent of Handler (2006. PN: U.S.Pat. No. 7,005,296B1).

DeVault disclose to use a female specific promoter to be ligated into alethal gene. The gene is only activated in females and therefore malesare uniquely remained in the selection. These males are irradiated forsterilization before releasing into nature (DeVault et al., 1996;Biotechnology, Vol 14; 46-49; DeVault et al.,1996. Genome Res. 6:571-579). This achievement gains a big progress in the genetic sexingexperiments, however the use of radiation can severely damage for smallinsects with its consequences of decreasing male mating competitivenessability.

To avoid the radiation damages, a new method named RIDL (Release ofInsects carrying a dominant lethal) has been disclosed in a patent(Alphey, 1999. PN: WO 01/39599 A2; Alphey, 2007. Area-Wide Control ofInsect Pests: From Research to Field Implementation, Springer,Dordrecht, The Netherlands). The RIDL offers a solution to many of thedrawbacks of traditional SIT that have limited its application inmosquitoes as mentioned above. RIDL differs from conventional SIT inthat the released insects are not sterilized by irradiation but itssterility is resulted from a homozygote for a dominant lethal gene.Highly efficient repressible RIDL systems were first demonstrated inDrosophila models and recently in the Mediterranean fruit fly (Thomas etal., 2000. Science, 287: 2474-2476.; Gong et al., 2000. Nature Biotech.,23: 453-456.). This system exploits a tetracycline-repressibletransactivator (tTA) to control expression of the dominant lethal(Gossen and Bujard, 1992. Proc Natl Acad Sci USA 1992,89(12):5547-5551). The tetracycline (Tet) that to be mixed in larvalrearing medium or food can bind to tTA and preventing it binding to tetOsequences and driving the effector gene. The tetO sequences plays a roleof an operator would be free to suppress the lethal gene. In the absenceof Tet, tTA protein binds to the operator sequence and the effector genewould be free to express. In natural environment where Tet is absent,released transgenic males mates with wild type females and theiroffspring would be killed by the effector gene activation. In Aedesmosquitoes, RIDL has been proved to be efficient, of which the malescreated haven't been declined their fitness when competing with wildtype males. (Phuc et al., 2007; BMC Biologyhttp://www.biomedcentral.com/1741-7007/5/11). However, the RIDL methodhas a serious shortcoming that it still produces offspring in bothsexes. It therefore needs an addition step of sexing to remove femalesbefore releasing.

Fu et al., (2010. PNAS, Vol. 107, No. 10, 4550-4554) discloses a methodin which a fusion between RIDL system and a female sex-specificregulation based on an endogenous Actin-4 promoter that derived fromAedes aegypti females. The effector gene is specifically activated onlyin the direct-flight muscle of female mosquitoes and this expressionmakes females to be flightless. These females after eclosion would bestuck on the water surface and to be dead eventually. By this method,only 50% of offspring becomes males which can continuously pass thetransformant genetic systems into next generation. However, this methodin practice has a shortcoming. This happens when plenty of theflightless females staying on the water surface, their bodies and legmovements can prevent other eclosion males to come up with the waterwhich may eventually drown males. The higher rearing density is thehigher “collateral damage” for males, but in industrial insectary,rearing at high density is the only option.

Transformer-2 gene has been seen as a key factor in combination with Trafor sex determination in different eukaryotes although it may involvedifferently in different taxa depending on evolutionary divergence.Fortier and Belote (2000. Genesis 26(4): 240-244), Salvemini et al(2009. Int. J. Dev. Biol. 53: 109-120) and Sarno et al (2010.http://www.biomedcentral.com/1471-2148/10/140) used the RNAi method toknock down the Tra-2 genes in Drosophila, Ceratitis Capitata andAnastrepha, respectively. The knockdown effect can convert females ofthese species into pseudo males carrying XX chromosomes. In theirstudies, the RNA interference method is performed by injection of Tra-2double stranded RNA (dsRNA) into embryos after an invitro synthesizedstep. No tra-2 orthologue has been identified in Anopheles and the Tra-2orthologue in Aedes aegypti mosquitoes seems to involve in a differentgenetic mode. A full length mRNA transcription of Tra-2 gene in Aedesaegypti is not necessarily required for its downstream gene cascade,doublesex (dsx) to be spliced. One female specific Dsx can be defaultspliced to be females (Salvemini et al., 2011. BMC Evolutionary Biology2011, 11:41 http://www.biomedcentral.com/1471-2148/11/41). It,therefore, suggests that the wish to create all maleness offspring,including 50% pseudo [XX (mm)] males by a conversion from females isimpossible if targeting Tra-2 in Culicinae. In fact, in our experiments,Tra-2 dsRNA injection into Culicinae mosquito eggs hasn't caused asignificant bias in sex ratio.

The present invention sets out to overcome all the shortcoming of theprevious methods by using the common principles of the RIDL method(Alphey, 1999. PN: WO 01/39599 A2; Alphey, 2007. Area-Wide Control ofInsect Pests: From Research to Field Implementation, Springer,Dordrecht, The Netherlands) in combination with a discovery of X (m)bearing sperm killing effect due to Tra-2 RNAi genetic system. Thesetransgenic Culicinae mosquitoes are therefore to produce more than 90%genetic maleness offspring.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 describes RNAi kernel sequences controlled by a regulatoryelement in a transactivator system within a PiggyBac plasmid. This a isdiagrammatic representation of a completed Tra2-RNAi system, linearizedat the 3′ end of the piggyBac transposon. There are three functionalsegments within the ends of the transposon: the marker (3xP3promoter-ECFP-SV40 poly A3′) to allow detection of transgenicindividuals by fluorescence. tTA protein is under the control of aninsect spermatogenesis promoter (Drosophila β2) and terminated by a SV40poly A 3′ sequence. The third segment is the Tra-2 Rnai cassette(tetO7-CMV minimal promoter—two Tra2 mRNA inverted repeats joined by afragment of a linker or intron sequence. This is also terminated by aSV40 poly A 3′ sequence. The Tra-2 Rnai cassette acts by a tTA proteinbinding into the tet07 in the absence of Tetra cycline to free CMVminimal promoter to drive.

SUMMARY AND TECHNIQUE PRINCIPLES OF THE INVENTION

We discovered that is not like the Tra-2 in Drosophila, CeratitisCapitata and Anastrepha, the Tra-2 gene in Culicinae mosquitoes isinvolved in male specific, spermatogenesis processes and the knockdownof Tra-2 gene hasn't resulted in a conversion from females to males asoccurred in the other Dipteran insects. Because, a transient effect ofdsRNA cannot last from eggs to adults to consistently cause a knockdowneffect into spermatogenesis stages and following consequences in nextgeneration, therefore all attempts to use dsRNA injections to obtaintransient effects would be invalid.

To successfully repress the Tra-2 gene in mosquitoes, it is necessary tocreate permanent transgenic lines with Tra-2 RNAi constructs by whichthe interference effect will be stably expressed during thespermatogenesis stages. We found that Tra-2 gene knockdown in Culicinaemosquitoes can cause lethality of X (m) chromosome bearing sperms andtherefore only Y-chromosome (M) bearing sperms are survival and malenessoffspring would be produced. These males are genetic males which carry aY chromosome. Males created by this method are not sterile but theyproduce healthy Y chromosome bearing sperms only. Theoretically,maleness offspring would continuously pass the Tra-2 RNAi constructsinto natural populations until the populations gone extinct.

This invention discloses all of the methods to create Tra-2 RNAi DNAconstructs, to transform it into mosquitoes and to observe itsexpression.

The invention uses putative Transformer-2 encoding gene sequences fromAedes albopictus, Aedes polynesiensis, Culex quinquefasciatus or theother Culicinae mosquitoes as materials to assemble Tra-2 RNAi geneticconstructs by using DNA recombination techniques. In Examples of thisinvention, parts or whole RRM (RNA recognition motif) sequences whichare belonging to putative Transformer-2 encoding sequences from Aedesalbopictus, Aedes polynesiensis and Culex quinquefasciatus are used.

The Tra-2 of Aedes aegypti is identified by blasting against the Aedesaegypti Genbank database with an input is the Drosophila Tra-2A aminoacid sequence. The outcome was AAEL004293-RA protein belonging tosupercontig 1.113 (Aedes aegypti-Vectorbase). Aedes aegypti are closelyrelated species with Aedes albopictus and Aedes polynesiensis, thereforeprimers derived from the AAEL004293-RA sequence can be used to amplifyTra-2 sequences of Aedes albopictus and Aedes polynesiensis mosquitoes.The regions with highest similarity among the orthologous Tra-2 genesare RRMs (RNA recognition motives) with a length of 240 bp. For manyother Aedes spp these primers were tested and can successfully amplifythese 240 bp regions. We found two RRMs loci (or allele), each exists inboth Aedes albopictus and Aedes polynesiensis. They are different 10%amino acid from each other and named as SEQ ID: No 1 (RRM1) and SEQ ID:No 2(RRM2). To knock down these two loci (allele), it may be required totransform two respective RNAi constructs into each species to repressthe respective RRM locus (allele).

A putative Tra-2 gene of Culex quinquefasciatus is identified byblasting Culex quinquefasciatus database with the RRM1 and RRM2sequences. The name of the outcome is CPIJ016646; supercontig3.780:5008-5247. The RRMs of Culex orthologous Tra-2 gene is named asSEQ ID: No 3 (RRM3). For many other Culex spp, primers derived from thefirst and the end of the RRM3 region have been tested and cansuccessfully amplify these 240 bp regions.

In order to knock down the Tra-2 genes in Aedes albopictus, Aedespolynesiensis, Culex quinquefasciatus and the other Culicinae mosquitoesby the RNAi technique, there are three solutions to be disclosed in theinvention.

-   -   The first solution is to use SEQ ID: No 1 (RRM1) as materials to        invitro create an RNAi kernel sequence 1. This is a recombinant        DNA fragment combining two identical sequences of RRM1 but in        opposite directions. A connection between the two RRM1 repeats        is a straight intron or linker DNA sequence. The RNAi kernel        sequence 1 is then ligated with transactivator and regulatory        elements and a fluorescent marker within a PiggyBack plasmid.        This plasmid would then be available for transforming into both        Aedes albopictus and Aedes polynesiensis to knock down their        RRM1 locus.

The regulatory element for the kernel sequence is a minimal promoterassociated with operator sequences (tetOx7). The minimal promoter plustetOx7 can be conditional by using the commercial transactivatorregulation systems (Clontech). Gene expression is activated as a resultof binding of the Tet-Off protein (tTA) to tetracycline responseelements (TREs) located within the minimal promoter.

For the best of expression, the invention suggests to use insectspermatogenesis specific promoters to control tTA protein gene. Besides,we also prefer to derive a minimal promoter from an insectspermatogenesis specific promoter for controlling the RNAi kernelsequence 1 which helps to eliminate all leakiness effect. This solutionmay be applied for any other Aedes spp which has a highly similarsequence of its RRM in comparison with the RMM1 or after obtaining itsown Tra-2 RRM sequences by the same pair of primers.

-   -   The second solution is to use SEQ ID: No 2 (RRM2) as materials        to invitro create an RNAi kernel sequence 2. This is a        recombinant DNA fragment combining two identical sequences of        RRM2 but in opposite directions. A connection between the two        RRM2 repeats is a straight intron or linker DNA sequence. The        RNAi kernel sequence 2 is then ligated with regulatory elements        and a fluorescent marker within a piggyBack plasmid. This        plasmid would then be available for transforming into both Aedes        albopictus and Aedes polynesiensis to knock down their RRM2        locus.

In the details, the regulatory elements in the second solution areidentical as those described in the first solution.

This solution may be applied for any other Aedes spp which has a highlysimilar sequence of its RRM in comparison with the RMM2 or afterobtaining its own Tra-2 RRM sequences by the same pair of primers.

-   -   The third solution is to use SEQ ID: No 3 (RRM3) as materials to        invitro create a RNAi kernel sequence 3. This is a recombinant        DNA fragment combining two identical sequences of RRM3 but in        opposite directions. A connection between the two RRM3 repeats        is a straight intron or linker DNA sequence. The RNAi kernel        sequence 3 is then ligated with regulatory elements and a        fluorescent marker within a piggyback plasmid. This plasmid        would then be available for transforming into Culex        quinquefasciatus.

In the details, the regulatory elements in the third solution areidentical as those described in the first and second solutions. Thissolution can be applied for any other Culex spp which has a highlysimilar sequence of its RRM in comparison with the RMM3 or afterobtaining its own Tra-2 RRM sequences by a pair of primers to bementioned in Examples.

The connective intron or linker between two Tra-2 RRM inverted repeatscan be any eukaryote intron sequence. However, introns from therespective species are preferred. The length of connective linker orintron can be from few to few hundred nucleotides. We prefer that twonucleotides of GT and AG need to be inserted at the beginning and at theend of intron or linker, respectively. These are the strengtheningsignals for spliceosomes to recognize and to splice out the connectiveintrons or linkers.

Three DNA Tra-2 RNAi RRM-1, Tra-2 RNAi RRM-2, Tra-2 RNAi RRM-3 kernelsequences are disclosed here as examples of using specifically Tra-2 DNAsequences to produce an interference effect into the respective species.The transcription of the DNA kernel structure containing two identicallyinverted repeats would produce single strands of mRNAs, which exposestwo complementary sequences at its two ends. The complementary sequenceswould bind together forming a double strand hairpin mRNA structure(dsRNAi), in which the looping part is formed by the intron or linker.Mosquito cells recognize the abnormal structure and react by dicing thedsRNA interference in a defend mechanism activity which is followed bydestroying intact single strand Tra-2 mRNAs from itself (Wang et al.,2006. Cell 127, 803-815; Hammond et al., 2001. Nat Rev Genet.2(2):110-9). The Tra-2 gene is, therefore, knocked down.

Illustrations and its Explanations.

The RNAI kernel seqence 1

Tra-2 RRM RNAi-1 Forward sequence Connection Reverse sequence5′AGTAAGTGCCTCGGTGTGTTCGGCCTAA GCAGCTACACCAACGAAACCAGCCTGATGGACGTTTTCGCACCGTACGGAACCATTGACA AGGCGATGATTGTCTACGATGCCAAGACGA AGGTTTCCCG

GGGTTCGGATTCGTGTACT TCCAGGAGCAGAGTGCGGCCACCGAAGCCA AAATGCAGTG

AATGG

ATGATGCTGCATG AGCGCACGATTAGAGTGGATTATTCGGTGA CC-3′ (SEQ ID NO: 1)GT-Intron-AG (or linker) 5′GGTCACCGAATAATCCACTCTAATCGTGCGCTCATGCAGCATCAT

CCATT

CACTGCATTTTGGCTTCGGTGGCCGCA CTCTGCTCCTGGAAGTACACGAATCCG AACCC

CGGGAAACCTTCGTCTTGGCA TCGTAGACAATCATCGCCTTGTCAATGGTTCCGTACGGTGCGAAAACGTCCATC AGGCTGGTTTCGTTGGTGTAGCTGCTTAGGCCGAACACACCGAGGCACTTACT-3′ (SEQ ID NO: 4)The respective amino acid sequence 1 -S--K--C--L--G--V--F--G--L--S--S--Y--T--N--E--T--S--L--M--D-21 -V--F--A--P--Y--G--T--I--D--K--A--M--I--V--Y--D--A--K--T--K-41 -V--S--R--G--F--G--F--V--Y--F--Q--E--Q--S--A--A--T--E--A--K-61 -M--Q--C--N--G--M--M--L--H--E--R--T--I--R--V--D--Y--S--V--T-(SEQ ID NO: 5) Note: 3′ end of the forward sequence is connected with anintron or linker sequence by GT. 5′ end of reverse sequence is connectedwith the intron or linker sequence by AG. The bold italicized charactersindicate substitutions. Y, S, N, K etc show different possibilities ofchanged nucleotides at the same position. The substitutions may or maynot change amino acid code. The RNAi kernel sequence 2.

Tra-2 RRM RNAi-2 Forward sequence Connection Reverse sequence5′AGTAAGTGCCTCGGTGTGTTCGGCCT

AG

AGCTA

ACCA

CGAA

CCA

CCTGAT GGA

GT

TTC

C

CCGT

CGG

ACCAT

G ACAAGGC

ATGATTGTCTACGATGCCAAG ACGAAGG

TCCCG

GGGTT

GG

TTCGT GTA

TTCCAGGAGCAGAGT

CGGCCAC

G AGCCAAA

TGCAGTG

AA

GGAATG

CTGCA

GAGCG

ACGATTAGAGTGGATTA TTCGGTGACC-3′ (SEQ ID NO: 2) GT-Intron- AG (or linker)5′GGTCACCGAATAATCCACTCTAATCGT

CGCTC

TGCAG

CATTCC

TT

CACTGCA

TTTGGC

T C

GTGGCCG

ACTCTGCTCCTGGAA

TACACGAA

CC

AACCC

CGGGA

CCTTCGTCTTGGCATCGTA GACAATCAT

GCCTTGTC

ATGGT

CCG

ACGG

G

GAA

AC

TCCATCAGG

TGG

TTCG

TGGT

TAG CT

CT

AGGCCGAACACACCGAGGCACTTACT-3′ (SEQ ID NO: 6)The respective amino acid sequence  1-S--K--C--L--G--V--F--G--L--S--S--Y--T--

--E--T--

--L--M--D- 21 -V--F--S--P--

--G--T--I--D--K--A--M--I--V--Y--D--A--K--T--K- 41-A--S--R--G--F--G--F--V--Y--F--Q--E--Q--S--

--A--T--E--A--K- 61 -

--Q--C--N--G--M--

--L--H--E--R--T--I--R--V--D--Y--S--V--T- (SEQ ID NO: 7) Note: 3′ end ofthe forward sequence is connected with an intron or linker sequence byGT. 5′ end of reverse sequence is connected with the intron or linkersequence by AG. The bold italicized characters indicate substitutions.Y, S, N, K etc show different possibilities of changed nucleotides atthe same position. The substitutions may or may not change amino acidcode.

Tra-2RRMRNAi-3 Forward sequence Connection Reverse sequence5′CGTAACGGAATAGTCCACCCGGATG GTTCGCTCGTGCATTACCATTCCGTTGCACTGCACCTTGGCTGCGGAAGCGTCC TCCAGGTTGACAAAGTACACGAATCCGAACCCGCGGGACGCCTTCGTCTTGGCA TCGTACACGATCTGCACCTTCTCGATCAATCCGAACCGGCCAAACACGGTCCTC AGGTCCGCCTCCTGGGTGTAATTGCTGAGGCCAAACACGCCGAGGCAGGTCGA-3′ (SEQ ID NO: 3) GT-Intron-AG (or linker)5′TCGACCTGCCTCGGCGTGTTTGGCCTC AGCAATTACACCCAGGAGGCGGACCTGAGGACCGTGTTTGGCCGGTTCGGATTGATCG AGAAGGTGCAGATCGTGTACGATGCCAAGACGAAGGCGTCCCGCGGGTTCGGATTCGT GTACTTTGTCAACCTGGAGGACGCTTCCGCAGCCAAGGTGCAGTGCAACGGAATGGTA ATGCACGAGCGAACCATCCGGGTGGACTATTCCGTTACG-3′ (SEQ ID NO: 8) The respective amino acid sequence 1 -S--T--C--L--G--V--F--G--L--S--N--Y--T--Q--E--A--D--L--R--T-21 -V--F--G--R--F--G--L--I--E--K--V--Q--I--V--Y--D--A--K--T--K-41 -A--S--R--G--F--G--F--V--Y--F--V--N--L--E--D--A--S--A--A--K-61 -V--Q--C--N--G--M--V--M--H--E--R--T--I--R--V--D--Y--S--V--T-(SEQ ID NO: 9) Note: 3′ end of the forward sequence is connected with anintron or linker sequence by GT. 5′ end of reverse sequence is connectedwith the intron or linker sequence by AG. The bold italicized charactersindicate substitutions. Y, S, N, K etc show different possibilities ofchanged nucleotides at the same position. The substitutions may or maynot change amino acid code.

DETAILED DESCRIPTION OF THE INVENTION

The Tra-2 RNAi system in the present invention may be any part of Tra-2encoding sequences (mRNA) of Tra-2 genes originated from Aedesalbopictus, Aedes polynesiensis, Culex quinquefasciatus or the otherCulicinae mosquitoes which are capable of producing a knockdown(interference) effect to the Tra-2 gene of the respective species. Wenot rule out the possibilities that a Tra-2 RNAi system containing Tra-2recombinant sequences from a certain Culicinae mosquito species can alsocause a knockdown (interference) effect to the other closely relatedmosquito species within Culicinae.

Definitions:

-   -   Culicinae mosquitoes refer to mosquito species which have a pair        of chromosome (chromosome I) that are similar in size but are        distinguishable in many species by the presence in the X (m) or        absence in the Y(M) of C-banding intercalary heterochromatin        (Knudson et al., 1996. 175-214. The Biology of Disease vectors.        University Press of Colorado).    -   Tra-2 gene sequences from Culicinae mosquitoes refer to mRNA        coding sequences only (Latchman, 1998. Gene regulation. A        eukaryotic perspective. 3^(rd) edition. Stanley Thornes        Publishers).    -   The RNAi kernel sequences refer to any recombinant DNA sequence        which includes two inverted repeats (IR) in conjunction by a        linker or intron sequence. Sequence of the IR is derived from        any part of Culicinae Tra-2 mRNA sequences.    -   We definite that Tra-2 RNAi kernel sequences is an RNAi encoding        sequence, its expression is under the control of a repressible        transactivator protein system.

As mentioned above, we look for an existence of the Tra-2 genes in Aedesalbopictus, Aedes polynesiensis and Culex quinquefasciatus mosquitoeswhich may contain a highly conserved region with a length of 240 bp (80amino acids). This region has been identified in Drosophila, CeratitisCapitata as Tra-2 RRM specific region (RNA recognition motif)http://www.expasy.orq/cqi-bin/prosite-search-ac?PDOC00030.

Using a sequence from 1221182 to 1220943 of Aedes aegypti Tra-2 gene(GenBank accession number: AAEL004293-RA), two primers have beendesigned. A forward is CLF, 26 bp at beginning of the RRM region(AGTAAGTGCCTCGGTGTGTTCGGCCT) (SEQ ID NO:10) and a reverse is CLR, 23 bpat the end of the RRM region (CCGGTCACCGAATAATCCACTCAA) (SEQ ID NO:11).PCR products amplified on DNA templates from Aedes albopictus and Aedespolynesiensis were sequenced. It revealed that in each species there aretwo different RRM sequences, RRM1 and RRM2. RT-PCR has shown expressionfrom both of those RRMs. RRM1 is identical with the Tra-2 RRM in Aedesaegypti (AAEL004293-RA) but RRM2 has 10% amino acid differences. Thesepair of primers can be used to amplify ortholog Tra-2 RRM 240 bp regionsfrom the other Aedes spp, even the distance species as Aedes niveus,Aedes annandalei or Aedes pseudoalbopictus. The amplification conditionis similar.

A Tra-2 sequence of Culex quinquefasciatus is available from Genbank(GenBank accession number: CPIJ016646) when using the RRM1 and RRM2sequence as inputs to blast. Its RRM sequence belongs to the supercontig3.780, from 5008 to 5247. We named it as RRM3. 24 bp at beginning and 22bp at the end of RRM3 are used to create a pair of primers to amplify itfrom Culex quinquefasciatus DNA. These pair of primers can be used toamplify ortholog Tra-2 RRM 240 bp regions of the other Culex spp, eventhe distance species as Culex visnue, Culex pipiens. The amplificationcondition is similar.

For convenience in designing primers whole or a part of the RRM regions(RRM1, RRM2 and RRM3) can be used to assemble the Tra-2 RNAi constructsby DNA recombinant techniques. However, in this invention, it doesn'tlimit to use other Tra-2 encoding parts outside the RRM region of thesemosquitoes to build other Tra-2 RNAi constructs.

The elements that regulate the RNAi kernel sequences should be locatedon the same chromosome as the RNAi kernel sequences. In FIG. 1 shows theRNAi kernel sequences and Tetracycline (Tet) transactivator system. Aninsect spermatogenesis promoter, for instance Drosophila β2, controlsthe tTA protein gene. In the presence of Tet in larva rearing medium,tTA protein binds to Tet and the operator sequence (tetO7) would bind tothe minimal promoter which regulates the transcription of the kernelstructure. The promoter is inactivated and no RNAi product istranscribed. In the absence of Tet, this artificial protein binds tooperator tetOx7 in the absence of Tetracycline (Tet) and the minimalpromoter would be free to transcribe the RNAi strand. In the sameplasmid, a reporter gene as ECFP, Dsred2 or EGFP can be ligated to a3xP3 or Actin5C promoter. We can trace the plasmid by following thisfluorescent marker. The entire packet is ligated into a PiggyBacplasmid. This complex can be transformed into mosquito geneticbackground in one or more loci which can be in the same or differentchromosomes.

We suggest that a single locus of the transgene in a transgenic line canbe used as a background for another transformation. A secondtransformant locus which occurs in the same chromosome with the firstone, would be particularly preferred. Transformants occurred in the samechromosome would prevent them to be segregated in the next generationsand especially in the case where the two Tra-2 loci (or allele) exist insame species as Aedes albopictus and Aedes polynesiensis, in which tworespective RNAi transformants are necessary to repress two loci (orallele).

The expression of the RNAi kernel sequences would knock down the Tra-2gene in the transgenic species. The knockdown effect results in lethal Xchromosome bearing sperms and only male offspring is outcome.

Examples

Components: 1/RRM Tra-2 sequences: In this examples, we used three typesof RRMs (RRM1, RRM2 and RRM3) to create Tra-2 RNAi kernel constructs.These sequences are obtained from sequencing the target species orblasting from (http://www.vectorbase.orq/). It doesn't limit to use adifferent part of the Tra-2 gene sequences which are belonging to Aedesalbopictus, Aedes polynesiensis, Culex quinquefasciatus or the otherCulicinae moquitoes in the invention. All the other components ofplasmids are identical. 2/Drosophila β2 tubulin promoter (or otherinsect spermatogenesis promoter): PCR from Drosophila DNA.3/Transactivator component (tTA): Clontech. 4/Regulator element(tetOx7): Clontech. 5/Reporter gene:http://piggybac.bio.nd.edu/.6/Piggybacplasmids:http://piagybac.bio.nd.edu/.7/Helperplasmid:http://piqqybac.bio.nd.edu/.

I. RRMs from Aedes Albopictus and Aedes Polynesiensis.

These examples show how the RRM sequences of Tra-2 were identified fromAedes albopictus and Aedes polynesiensis. It also shows the way tocreate the RNAi kernel sequence by using the RRM sequences from Aedesalbopictus and Aedes polynesiensis.

As mentioned above, RRM regions of Aedes albopictus and Aedespolynesiensis have been amplified by a PCR used a pair of primers of 26bp at beginning and 23 bp at the ending of Aedes aegypti supercontig1.113 (1221182-1220943).

(SEQ ID NO: 10) CLF AGTAAGTGCCTCGGTGTGTTCGGCCT (SEQ ID NO: 11) CLRCCGGTCACCGAATAATCCACTCAADNA from Aedes albopictus and Aedes polynesiensis are extracted using aQIAGEN kit. PCR is carried out in 25 μl reaction in a condition of 2.5μl PCR buffer; 1.5 μl MgCL (25 mM); 0.5 μl dNTPs (10 mM); each primer0.5 μl (10 pmol/μl); 0.15 μl Taq DNA polymerase (5U/μl); 10-40 ng DNAtemplate. Thermal profile of PCR is [94oC/4; (94oC/30″; 59oC/30″;72oC/45″)×3; (94oC/30″; 57oC/30″; 72oC/45″)×3; (94oC/30″; 54oC/30″;7200/45″)×35; 72oC/10]. PCR products are then purified and sequencedwith the same primers. Two 240 bp sequences of RRMs are obtained below.

RRM1 DNA sequence (SEQ ID NO: 1)5′AGTAAGTGCCTCGGTGTGTTCGGCCTAAGCAGCTACACCAACGAAACCAGCCTGATGGACGTTTTCGCACCGTACGGAACCATTGACAAGGCGATGATTGTCTACGATGCCAAGACGAAGGTTTCCCG

GGGTTCGGATTCGTGTACTTCCAGG AGCAGAGTGCGGCCACCGAAGCCAAAATGCAGTG

AATGG

ATGATGCTGCATGAGCGCACGATTAGAGTGGATTATT CGGTGACC-3′.Underlined regions disclosed as SEQ ID NOS 10, 12 and 13, respectively, inorder of appearance. RRM2 DNA sequence (SEQ ID NO: 2)5′AGTAAGTGCCTCGGTGTGTTCGGCCT

AG

AGCTA

ACCA

CGAA

CCA

CCTGATGGA

GT

TTC

C

CCGT

CGG

ACCAT

GACAAGGC

ATGATTGTCTACGATGCCAAGACGAAGG

TCCCG

GGGTT

GG

TTCGTGTA

T TCCAGGAGCAGAGT

CGGCCAC

GA

GCCAAA

TGCAGTG

AA

GGAATG

CTGCA

GAGCG

ACGATTAGAG TGGATTATTCGGTGACC-3′Underlined regions disclosed as SEQ ID NOS 10, 14 and 13, respectively, inorder of appearance. RRM1 amino acid sequence (SEQ ID NO: 5) 1 -S--K--C--L--G--V--F--G--L--S--S--Y--T--N--E--T--S--L--M--D-21 -V--F--A--P--Y--G--T--I--D--K--A--M--I--V--Y--D--A--K--T--K-41 -V--S--R--G--F--G--F--V--Y--F--Q--E--Q--S--A--A--T--E--A--K-61 -M--Q--C--N--G--M--M--L--H--E--R--T--I--R--V--D--Y--S--V--T-RRM2 amino acid sequence (SEQ ID NO: 7) 1 -S--K--C--L--G--V--F--G--L--S--S--Y--T--

--E--T--

--L--M--D- 21 -V--F--

--P--

--G--T--I--D--K--A--M--I--V--Y--D--A--K--T--K- 41 -

--S--R--G--F--G--F--V--Y--F--Q--E--Q--S--

--A--T--E--A--K- 61 -

--Q--C--N--G--M--

--L--H--E--R--T--I--R--V--D--Y--S--V--T-(Underlines indicate the region selected for primers. Bold italics indicatenucleotide and amino acid substitutions).Beside, these pair of primers can be used to amplify this Tra-2 RRM 240bp region of the other Aedes spp, even from the distance species asAedes niveus, Aedes annandalei or Aedes pseudoalbopictus. Using the samePCR condition, an exact 240 bp band would be amplified among otherbands. An agarose gel extraction step is performed for the 240 bp bandby Qiagen columns. The DNA elution is diluted between 10-20 times inwater and 1 μl to be used as template for the same PCR. A 240 bpspecific band would be amplified and can be used to assemble Tra-2 RNAiconstructs for the respective species.

Two fragments of 135 bp from the bottom parts of these RRM1 and RRM2regions are used to assemble Tra-2 RNAi constructs. Because thesequences of RRM1 and RRM2 are only different in some parts, thereforethe primers derived outside of those parts can be used for amplifyingboth RRMs. PCR is carried out in 25 μl reaction in a condition of 2.5 μlPCR buffer; 1.5 μl MgCL (25 mM); 0.5 μl dNTPs (10 mM); each primer 0.5μl (10 pmol/μl); 0.15 μl Taq DNA polymerase (5U/μl); 10-40 ng DNAtemplate. Thermal profile of PCR is [94oC/4; (94oC/30″; 59oC/30″;72oC/45″)×3; (94oC/30″; 57oC/30″; 72oC/45″)×3; (94oC/30″; 54oC/30″;72oC/45″)×35; 72oC/10′]

1-(BA-EX1F) (SEQ ID NO: 15) 5′CGATCTCGGATCCATGCCAAGACGAAGGTTTCCCGAG 3′2-(X-Ex1R) (SEQ ID NO: 16)5′CGGCAATGACCTCGAGACCGGTCACCGAATAATCCACTCAA 3′ 3-(SAL-EX1F)(SEQ ID NO: 17) 5′GGCGTCAATGTCGACATGCCAAGACGAAGGTTTCCCGAG 3′4-(ECORI-EX1R) (SEQ ID NO: 18)5′CGGACGTTGGAATTCGACGGTCACCGAATAATCCACTCAA 3′

Primers 1 & 3 or 2 & 4 are similar forward and reverse primers. Acombination between 1 & 2 would produce the same PCR product as that of3 & 4. The differences in those PCR products are endonucleaserestriction enzyme sequences inserted in the front parts of the primers(underline). This allows the PCR products can be ligated to an intron orlinker that contains the same restriction sites in a desired direction.If a connection between the two inverted repeats is a linker about 10bp, PCRs to amplify these fragments can use the same reverse primer (2or 4) and therefore products would contain the same restriction sites at3′ end (XhoI or EcoRI). Two PCR products would be easily inverselyconnected after an XhoI or EcoRI enzyme treatments. However, if we wantto insert an intron between the two inverted repeats, it needs to useboth inverse primers. Two PCR products would then have different stickyends at 3′ (XhoI and EcoRI) and can be easily ligated with an intronthat ends by XhoI and EcoRI restriction sites. In this invention, anylinker or intron sequence from other insects can be used in conjunctionwith the two inverted repeats, provided that two nucleotides GT and AGwould be inserted at the first and the end of those sequences,respectively. These are recognition signals for intron splicing sites.

After two identical DNA fragments are reversely connected via an intronor linker, these RNAi kernel sequences (1 & 2) can be easily ligatedinto the transactivator system in a desired direction provided that thetransactivator plasmids contain the same restriction sites.

II. RRM from Culex Quinquefasciatus

Genomic sequences of Culex quinquesfaciatus are available in theVectorbase.org website (http://www.vectorbase.org/). We used two RRMs(RRM1 and RRM2) as queries to blast against the database. Outcome is a240 bp sequence which is highly similar with RRM1 and RRM2 in its helixstructure as well as phylogenic relationship. RRM3 contains up to 69%and 73% amino acid similarity with RRM1 and RRM2, respectively. Theannotation of Tra-2 Culex quinquesfaciatus is CPIJ016646;supercont3.780:5008-5247.(RRM3)

RRM3 DNA sequence (SEQ ID NO: 3)CGTAACGGAATAGTCCACCCGGATGGTTCGCTCGTGCATTACCATTCCGTTGCACTGCACCTTGGCTGCGGAAGCGTCCTCCAGGTTGACAAAGTACACGAATCCGAACCCGCGGGACGCCTTCGTCTTGGCATCGTACACGATCTGCACCTTCTCGATCAATCCGAACCGGCCAAACACGGTCCTCAGGTCCGCCTCCTGGGTGTAATTGCTGAGGCCAAACACGCCGAGGCAGGTCGA.Underlined regions disclosed as SEQ ID NOS 19 and 20, respectively,in order of appearance. RRM3 amino acid sequence (SEQ ID NO: 9) 1 -T--C--L--G--V--F--G--L--S--N--Y--T--Q--E--A--D--L--R--T-21 -V--F--G--R--F--G--L--I--E--K--V--Q--I--V--Y--D--A--K--T--K-41 -A--S--R--G--F--G--F--V--Y--F--V--N--L--E--D--A--S--A--A--K-61 -V--Q--C--N--G--M--V--M--H--E--R--T--I--R--V--D--Y--S--V--T-(Underlines indicate the region selected for primers).

In Culex quinquesfaciatus whole RRM3 sequence can be used to create anRNAi kernel sequence as its nucleotide sequences at beginning and at theend are suitable to design good primers. 24 bp at beginning and 22 bp atthe end of RRM3 (underline) are used to create a pair of primers. Thesepair of primers can be used to amplify this Tra-2 RRM 240 bp region ofthe other Culex spp, even the distance species as Culex vishnue, Culexpipiens or Culex tritaeniorhynchus by a similar condition. Using thesame KR condition, an exact 240 bp band would be amplified among otherbands. A gel extraction step is performed for the 240 bp band by Qiagencolumns. The DNA elution is diluted between 10-20 times in water and 1μl to be used as template for the same PCR. A 240 bp specific band wouldbe amplified and can be used to assemble Tra-2 RNAi constructs for therespective species.

7-(BA-EX1F) (SEQ ID NO: 21) 5′CGATCTCGGATCCCGTAACGGAATAGTCCACCCGGAT 3′8-(X-Ex1R) (SEQ ID NO: 22) 5′CGGCAATGACCTCGAGACTCGACCTGCCTCGGCGTGTTTG 3′9-(SAL-EX1F) (SEQ ID NO: 23)5′GGCGTCAATGTCGACCGTAACGGAATAGTCCACCCGGAT 3′ 10-(ECORI-EX1R)(SEQ ID NO: 24) 5′CGGACGTTGGAATTCGATCGACCTGCCTCGGCGTGTTTG 3′

PCR is carried out in 25 μl reaction in a condition of 2.5 μl PCRbuffer; 1.5 μl MgCL (25 mM); 0.5 μl dNTPs (10 mM); each primer 0.5 μl(10 pmol/μl); 0.15 μl Taq DNA polymerase (5U/μl); 10-40 ng DNA template.Thermal profile of PCR is [94oC/4; (94oC/30″; 59oC/30″; 7200/45″)×3;(94oC/30″; 57oC/30″; 72oC/45″)×3; (94oC/30″; 54oC/30″; 7200/45″)×35;72oC/10′]. Afterward, these PCR products are also performed in the samemanner with those have been done in Aedes albopictus and Aedespolynesiensis. Whatever, these fragments are connected by a linker orintron, after this RNAi kernel sequence (3) is constructed, it would beavailable to ligate into the transactivator plasmids to transform Culexquinquesfaciatus embryos.

III. Connection of the RNAi Kernel Structures with Tre Repressor.

The pTre-tight plasmid (Cat. No. 631059) from Clontech is mixed with theRNAi kernel sequence (1 or 2 or 3) in 1:3 molar ratio in a 30 μlreaction in the presence of BamHI and SaII restriction enzymes. Afterdigestion, ligation is performed by adding T4 ligation into thedenatured restriction enzyme mixture. The circle plasmid is transformedinto competent cells (DH5α™ derivative, New England Biolabs), isolatedand cultured overnight to harvest a larger amount of plasmid DNA fromeach clone. The size of new plasmid would be 2.6 kb plus the size of theRNAi kernel sequences. In the case of RRM1 and RRM2 from Aedesalbopictus and Aedes polynesiensis, only 135 bp of each RRM are used,the plasmid size would be about 3070 bp if using an intron of 200 bp. Ifa linker of 10 bp is used, the plasmid is about 2870 bp. In the case ofCulex quinquesfaciatus, whole 240 bp is used, if it is accompanied with200 bp intron, the fragment size would be 3280 bp. If a linker of 10 bpis used, the plasmid is about 3090 bp.

A fragment includes the Tre operator and the RNAi kernel sequence(tetOx7+PminCMV+RNAi kernel sequence +SV40 polyA) can now be amplifiedby two primers which contain HindIII and Acc65I restriction sites. Thesepending sites are available for ligation with Piggybac plasmid and theother parts of the construct.

(Tre-HindIII) (SEQ ID NO: 25) CGATCTAAGCTTCTCGAGTTTACTCCCTATCAGTGA(Tre-Acc65I) (SEQ ID NO: 26) CGATCTGGTACCAGTCAGTGAGCGAGGAAGCTCGAG

PCR is carried out in 25 μl reaction in a condition of 2.5 μl PCRbuffer; 1.5 μl MgCL (25 mM); 0.5 μl dNTPs (10 mM); each primer 0.5 μl(10 pmol/μl); 0.15 μl Taq DNA polymerase (5 U/μl); 10-40 ng DNAtemplate. Thermal profile of PCR is [94oC/4; (94oC/90″; 54oC/60″; 72oC/2min 30″)×35; 72oC/10]. The PCR products amplified from the RRMs of Aedesalbopictus and Aedes polynesiensis with a 10 bp linker would be 875 bp,meanwhile a 200 bp intron would produced 1065 bp products. The PCRproducts amplified from the RRM of Culex quinquesfaciatus would be 1085bp and 1275 bp, which are respectively to a 10 bp linker or 200 bpintron. The PCR products are digested by Acc65I and HindIII and purifiedby Qiagen columns. The product is available for a final ligation.

IV. Connection of the Drosophila β2 Tubulin Promoter with aTransactivator Sequence.

Drosophila β2 tubulin promoter sequence is obtained from GenBank orhttp://flybase.org/reports/FBqn0003889.html. Two primers which containEcoRI and Apa I are designed from the sequence. These primers amplify230 bp of 5′UTR of β2 tubulin gene from Drosophila genomic DNA. Thermalprofile of PCR is [94oC/4; (94oC/30″; 55oC/30″; 72oC/45″)×35; 72oC/10].

β2-ApaI-F (SEQ ID NO: 27) CGATCTGGGCCCGGAAATCGTAGTAGCCTATTTGTGAβ2-EcoRI-R (SEQ ID NO: 28) CGGACGTTGGAATTCCCTGAATGTGTACAATTTCACGCAT

The pTet-Off-Advanced plasmid (Clontech, Catalog Nos. 630934) isdigested by two restriction enzymes EcoRI and HindIII producing a bandof 1222 bp. This DNA fragment is then ligated to the β2 tubulin promotersequence via the EcoRI restriction site to produce a fragment of 1458bp. tTA protein is now controlled by β2 tubulin promoter. The ligationproduct is digested by ApaI and purified by Qiagen columns. The productis available for a final ligation.

V. Whole Plasmid Assembles.

pXL-BacII-ECFP plasmid from http://piggybac.bio.nd.edu/ is used toassemble all the above fragments into completed Tra-2 RNAi constructs.The pXL-BacII-ECFP plasmid carries a 3xP3 promoter which drives ECEPreporter gene. This reporter gene would be tissue specific expressedunder the promoter. When mosquitoes are transformed with this marker,mosquito eyes would be fluorescently cyan color.

The pXL-BacII-ECFP plasmid is digested by ApaI and Acc65I and purifiedby Qiagen columns. The linear plasmid is 5390 bp. The plamid is thenmixed with Tre fragments (III), β2 +tTA fragment (IV) in 1:3:3 molarratio. T4 ligation is added into a 30 μl reaction.

Ligation product is used to transform into competent cells. Ligationproducts are expected in a range of different sizes as follow:

For Aedes albopictus and Aedes polynesiensis, two plasmid containing 10bp linker or 200 bp intron are 7723 bp and 7913 bp, respectively.Meanwhile, plasmids of Culex quinquesfaciatus would be 7933 bp and 8123bp for 10 bp linker and 200 bp intron, respectively.

VI. Plasmid Injection and Transformant Selection.

The Tra-2 RNAi plasmids is mixed with a pBSII-1E1-orf(http://piggybac.bio.nd.edu/) helper plasmid. The helper producestransposase enzyme which helps Piggybac in the Tra-2 RNAi plasmidsjumping into mosquito genome. A good concentration of the injectionmixture would be 600 ng of the Tra-2 RNAi plasmid plus 400 ng of thehelper per micro liter (μl) of 1× phosphate buffer. Mosquito eggs wouldbe injected within 2 hrs after oviposition into egg posterior ends.After 4 days post injection, the eggs are submerged into tetracyclinesolution (0.008 g per litter). Go survivors would be kept to cross withwild type males or females. G1 larvae are screened under a stereofluorescent microscope. Any fluorescent larva found that would be thetransformant one and to be crossed to build transformant lines. Theselines would be tested in Tet-on and Tet-off conditions to check sexratio. Any line having maleness skew over 80% in Te-off condition wouldbe kept for further analysis and for vector control applications.

Invention Effects

The method exposed in this invention would help to produce one sex(maleness) in Culicinae mosquitoes. Males created by this inventionwould pass on the Tra-2 RNAi genetic system into natural population whenbeing released. If the number of released males is big enough, it canresult in a collapse of natural vector population, even gone extinct ofwhole population in a certain time.

1. A method to make a Tra-2 RNAi kernel sequence, which method comprisesthe steps of: i) amplifying an RNA recognition motive (RRM) DNA sequencefrom a Culcinae mosquito selected from the group consisting of Aedesaldopictus, Aedus aegypti, Aedes polynesiensis and Culexquinquefasciatus, to obtain a first RRM DNA sequence of 240 base pairs,ii) repeating step i), to obtain a second RRM DNA sequence of 240 basepairs; iii) reversely connecting the first RRM DNA sequence with thesecond RRM DNA sequence via an intron or a linker DNA sequence, whichintron or linker DNA sequence is connected to the end of the first RRMDNA sequence and the beginning of the second RRM DNA sequence, so thatthe second RRM DNA sequence forms an inverted sequence to the first RRMDNA sequence, wherein the transcription of the first RRM DNA sequenceand the second RRM DNA sequences produces single strands of mRNA withcomplementary sequences exposed at ends of the strands of mRNA, whichcomplementary sequences are of sufficient length so as to be capable ofbinding together to form a double strand hairpin mRNA structure having aloop portion, where the loop portion is formed by the transcriptionproduct of the intron or linker DNA sequence.
 2. The method of claim 1,wherein the RRM DNA sequence is selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
 3. The method of claim 1,wherein the intron or linker DNA sequence is connected to the end of thefirst RRM DNA sequence and the beginning of the second RRMA sequence viaan AG sequence.
 4. A method to make a Tra-2 RNAi construct, which methodcomprises the steps of: i) creating an RNAi kernel sequence byamplifying an RNA recognition motive (RRM) DNA sequence from a Culcinaesequence selected from the group consisting of Aedes aldopictus, Aedusaegypti, Aedes polynesiensis and Culex quinquefasciatus, to obtain afirst RRM DNA sequence of 240 base pairs, amplifying an RNA recognitionmotive (RRM) DNA sequence from a Culcinae sequence selected from thegroup consisting of Aedes aldopictus, Aedus aegypti, Aedes polynesiensisand Culex quinquefasciatus, to obtain a second RRM DNA sequence of 240base pairs, reversely connecting the first RRM DNA sequence with thesecond RRM DNA sequence via an intron or a linker DNA sequence, whichintron or linker DNA sequence is connected to the end of the first RRMDNA sequence and the beginning of the second RRM DNA sequence, so thatthe second RRM DNA sequence forms an inverted sequence to the first RRMDNA sequence, wherein the transcription of the first RRM DNA sequenceand the second RRM DNA sequences produces single strands of mRNA withcomplementary sequences exposed at ends of the strands of mRNA, whichcomplementary sequences are of sufficient length so as to be capable ofbinding together to form a double strand hairpin mRNA structure having aloop portion, where the loop portion is formed by the transcriptionproduct of the intron or linker DNA sequence, ii) inserting atetracycline repressible transactivator operably linked to andcontrolling expression of the RNAi kernel sequence, and iii) insertingan insect spermatogenesis promoter operably linked and controllingexpression of the tetracycline repressible transactivator.
 5. The methodof claim 4, wherein the RRM DNA sequence is selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
 6. The method ofclaim 4, wherein the intron or linker DNA sequence is connected to theend of the first RRM DNA sequence and the beginning of the second RRMAsequence via an AG sequence.
 7. The method of claim 4, wherein theinsect spermatogenesis promoter is Drosophila-β2.
 8. The method of claim4, which comprises the further step of inserting a regulatory component,or a terminator component, or both.
 9. The method of claim 8, whereinthe regulatory component is tetOx7 and the terminator component is SV40polyA.
 10. A method for increasing genetic maleness offspring in aCulicinae mosquito population, comprising the steps of: producing aTra-2 RNAi construct, which method comprises the steps of: a) creatingan RNAi kernel sequence by i) amplifying an RNA recognition motive (RRM)DNA sequence from a Culcinae sequence selected from the group consistingof Aedes aldopictus, Aedus aegypti, Aedes polynesiensis and Culexquinquefasciatus, to obtain a first RRM DNA sequence of 240 base pairs,ii) amplifying an RNA recognition motive (RRM) DNA sequence from aCulcinae sequence selected from the group consisting of Aedesaldopictus, Aedus aegypti, Aedes polynesiensis and Culexquinquefasciatus, to obtain a second RRM DNA sequence of 240 base pairs,iii) reversely connecting the first RRM DNA sequence with the second RRMDNA sequence via an intron or a linker DNA sequence, which intron orlinker DNA sequence is connected to the end of the first RRM DNAsequence and the beginning of the second RRM DNA sequence, so that thesecond RRM DNA sequence forms an inverted sequence to the first RRM DNAsequence, wherein the transcription of the first RRM DNA sequence andthe second RRM DNA sequences produces single strands of mRNA withcomplementary sequences exposed at ends of the strands of mRNA, whichcomplementary sequences are of sufficient length so as to be capable ofbinding together to form a double strand hairpin mRNA structure having aloop portion, where the loop portion is formed by the transcriptionproduct of the intron or linker DNA sequence, iv) inserting atetracycline repressible transactivator operably linked to andcontrolling expression of the RNAi kernel sequence, and v) inserting aninsect spermatogenesis promoter operably linked and controllingexpression of the tetracycline repressible transactivator, b) stablytransforming a Culicinae mosquito with the Tra-2 RNAi DNA construct, c)allowing stable expression of the Tra-2 RNAi DNA construct duringspermatogenesis in the Culicinae mosquito, so as to effect Tra-2 geneknockdown of X(m) chromosome-bearing sperm, d) allowing the Culicinaemosquito of step ii) to mate and thereby stably pass on the Tra-2 RNAiDNA construct to offspring, resulting in continuous interruption of thedevelopment of X(m) chromosome-bearing sperm and thereby effectinggenetic male bias in progeny.
 11. The method for increasing geneticmaleness offspring in a Culicinae mosquito population of claim 10,wherein genetic male bias in progeny is at least 90%.
 12. A Tra-2 RNAiDNA construct produced by the method of claim
 4. 13. A Culicinaemosquito stably transformed with the Tra-2 RNAi DNA construct of claim12, wherein expression of the DNA construct occurs duringspermatogenesis so as to effect Tra-2 gene knockdown of X(m)chromosome-bearing sperms.